.gamma.-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the vertebrate brain, in the insect central nervous system and at insect neuromuscular junctions (Enna et al., 1986, In Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Alan R. Liss, Inc., New York, pp. 41-56; and Sattelle, D. B., 1990, Adv. Insect Physiol., vol. 22, pp. 1-113). Mammalian genes encoding at least 5 distinct classes of GABA receptor subunits, i.e., .alpha., .beta., .gamma., .delta., and .rho., have been cloned and characterized (Burr et al., GABA.sub.A receptor subtypes: from pharmacology to molecular biology, 1991, FASEB/J., vol. 5, 2916-2923). Genes encoding vertebrate GABA receptor subunits and the strychnine-binding subunit of the glycine-gated chloride channel comprise a family of homologous ligand-gated chloride channel genes, which is part of a larger superfamily of ligand-gated ion channel genes (Barnard et al., 1987, Trends Neurosci., vol. 10, pp. 502-509). Gene products of this superfamily have a conserved structural organization with four hydrophobic membrane-spanning domains. GABA stimulates chloride ion conductance through the associated chloride ion channel. The predominant effect of GABA is the interaction with a specific receptor protein which results in an increase of chloride ion conductance to produce an inhibition of neuronal firing. Heterologous expression in vitro of different combinations of GABA receptor subunit types (.alpha., .beta., .gamma., .delta., etc.) and subunit isoforms (.alpha.1, .alpha.2, etc.) results in heteromultimeric receptors with differing structure and pharmacology (Schofield, P. R., The GABA.sub.A receptor: molecular biology reveals a complex picture, 1989, Trends Pharmacol. Sci., vol. 10, pp. 476-478; and Burt et al., GABA.sub.A receptor subtypes; from pharmacology to molecular biology, 1991, FASEB/J., vol. 5, 2916-2923). It is thought that the differential expression of subunits in different cell types is the molecular basis of the known pharmacological diversity of GABA receptors in the mammalian central nervous system (Burt et al., 1991, cited elsewhere herein).
Electrophysiological studies of ligand-gated ion currents in insect nerve and muscle cells provide evidence for the existence of chloride channels gated by glutamate, histamine, and taurine, as well as those gated by GABA (Sattelle, D. B., 1990, cited elsewhere herein; and Lummis et al., 1990, Annu. Rev. Entomol., vol. 35, pp. 345-377). Although these findings imply the existence of a large and diverse gene family encoding ligand-gated chloride channels in insects, very little is known of about homologous channels of invertebrates. Only a single gene from insects having a significant degree of structural conservation and amino acid sequence identity with vertebrate ligand-gated chloride channel genes has been described (ffrench-Constant et al., 1991, Proc. Natl. Acad. Sci. USA, vol. 88, pp. 7209-7213). Here, a Drosophila melanogaster cDNA having significant predicted amino acid sequence identity to vertebrate ligand-gated chloride channel genes was isolated and mapped to a genetic locus (Rdl) that confers resistance to cyclodiene insecticides and other blockers of GABA-gated chloride channels. Rdl was shown to encode a GABA receptor subunit by the expression of functional homomultimeric GABA receptors in Xenopus oocytes following injection with Rdl mRNA (ffrench-Constant et al., 1993, Nature, vol. 363, pp. 449-451).
The only other example of a ligand-gated chloride channel gene from an invertebrate species is a GABA receptor .beta.-like subunit gene isolated from the pond snail, Lymnaea stagnalis (Harvey et al., 1991, EMBO/J., vol. 10, pp. 3239-3245). The functional relationship of the product encoded by this gene to vertebrate GABA receptor .beta. subunits was corroborated by the formation of a functional chimeric receptor with properties similar to vertebrate .alpha./.beta. heteromultimers when the gene was co-expressed with a vertebrate .alpha. subunit in Xenopus oocytes.
The .gamma.-aminobutyric acid (GABA) receptor-chloride channel complex mediates synaptic inhibition in both vertebrate and invertebrate nervous systems and is a target site for a variety of drugs, toxicants, and insecticides (Enna et al., 1986 and Sattelle, 1990, cited elsewhere herein). That is, GABA receptors of insects are known to be target sites for chemical agents having insecticidal or pesticidal activity. Furthermore, invertebrate GABA receptors have different pharmacological properties as compared to those of vertebrate GABA receptors. Therefore, the characterization and isolation of an invertebrate GABA receptor subunit gene(s) would be useful in developing screening techniques to identify insect-specific pesticides.